Immunomodulatory compounds

ABSTRACT

The present invention relates to novel heterocyclic compounds, to methods for their preparation, to compositions containing them, and to methods and use for clinical treatment of medical conditions which may benefit from immunomodulation, including rheumatoid arthritis, multiple sclerosis, diabetes, asthma, transplantation, systemic lupus erythematosis and psoriasis. More particularly the present invention relates to novel heterocyclic compounds, which are CD80 antagonists capable of inhibiting the interactions between CD80 and CD28.

[0001] The present invention relates to novel heterocyclic compounds, tomethods for their preparation, to compositions containing them, and tomethods and use for clinical treatment of medical conditions which maybenefit from immunomodulation, including rheumatoid arthritis, multiplesclerosis, diabetes, asthma, transplantation, systemic lupuserythematosis and psoriasis. More particularly the present inventionrelates to novel heterocyclic compounds, which are CD80 antagonistscapable of inhibiting the interactions between CD80 and CD28.

BACKGROUND OF THE INVENTION

[0002] The immune system possesses the ability to control thehomeostasis between the activation and inactivation of lymphocytesthrough various regulatory mechanisms during and after an immuneresponse. Among these are mechanisms that specifically inhibit and/orturn off an immune response. Thus, when an antigen is presented by MHCmolecules to the T-cell receptor, the T-cells become properly activatedonly in the presence of additional co-stimulatory signals. In theabsence of accessory signals there is no lymphocyte activation andeither a state of functional inactivation termed anergy or tolerance isinduced, or the T-cell is specifically deleted by apoptosis. One suchco-stimulatory signal involves interaction of CD80 on specialisedantigen-presenting cells with CD28 on T-cells, which has beendemonstrated to be essential for full T-cell activation. (Lenschow etal. (1996) Annu. Rev. Immunol., 14, 233-258)

[0003] A paper by Erbe et al, in J. Biol. Chem. Vol. 277, No. 9, pp7363-7368 (2002), describes three small molecule ligands which bind toCD80, and inhibit binding of CD80 to CD28 and CTLA4. Two of thedisclosed ligands are fused pyrazolones of structures A and B:

DESCRIPTION OF THE INVENTION

[0004] According to the present invention there is provided a compoundof formula (I) or a pharmaceutically or veterinarily acceptable saltthereof:

[0005] wherein

[0006] R₁ and R₃ independently represent H; F; Cl; Br; —NO₂; —CN; C₁-C₆alkyl optionally substituted by F or Cl; or C₁-C₆ alkoxy optionallysubstituted by F;

[0007] R₂ represents H, or optionally substituted C₁-C₆ alkyl, C₃-C₇cycloalkyl or optionally substituted phenyl;

[0008] Y represents —O—, —S—, N-oxide, or —N(R₅)— wherein R₅ representsH or C₁-C₆ alkyl;

[0009] X represents a bond or a divalent C₁-C₆ alkylene radical;

[0010] R₄ represents —C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆,—NHC(═O)NHR₆, or —NHC(═S)NHR₆ wherein

[0011] R₆ represents H, or a radical of formula -(Alk)b-Q wherein b is 0or 1, and

[0012] Alk is an optionally substituted divalent straight chain orbranched C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene or C₂-C₁₂ alkynylene radicalwhich may be interrupted by one or more non-adjacent —O—, —S— or —N(R₈)—radicals wherein R₈ represents H or C₁-C₄ alkyl, C₃-C₄ alkenyl, C₃-C₄alkynyl, or C₃-C₆ cycloalkyl, and

[0013] Q represents H; —CF₃; —OH; —SH; —NR₈R₈ wherein each R₈ may be thesame or different; an ester group; or an optionally substituted phenyl,C₃-C₇ cycloalkyl, C₅-C₇ cycloalkenyl or heterocyclic ring having from 5to 8 ring atoms; and

[0014] R₇ represents H or C₁-C₆ alkyl; or when taken together with theatom or atoms to which they are attached R₆ and R₇ form an optionallysubstituted heterocyclic ring having from 5 to 8 ring atoms.

[0015] Compounds of general formula (I) are CD80 antagonists. Theyinhibit the interaction between CD80 and CD28 and thus the activation ofT cells, thereby modulating the immune response.

[0016] Accordingly the invention also includes:

[0017] (i) a compound of formula (I) or a pharmaceutically orveterinarily acceptable salt thereof for use in the treatment ofconditions which benefit from immunomodulation.

[0018] (ii) the use of a compound of formula (I) or a pharmaceuticallyor veterinarily acceptable salt thereof in the manufacture of amedicament for the treatment of conditions which benefit fromimmunomodulation,.

[0019] (iii) a method of immunomodulation in humans and non-humanprimates, comprising administration to a subject in need of suchtreatment an immunomodulatory effective dose of a compound of formula(I) or a pharmaceutically or veterinarily acceptable salt thereof.

[0020] (iv) a pharmaceutical or veterinary composition comprising acompound of formula (I) or a pharmaceutically or veterinarily acceptablesalt thereof together with a pharmaceutically or veterinarily acceptableexcipient or carrier.

[0021] Conditions which benefit from immunomodulation include:

[0022] Adrenal insufficiency

[0023] Allergic angiitis and granulomatosis

[0024] Amylodosis

[0025] Ankylosing spondylitis

[0026] Asthma

[0027] Autoimmune Addison's disease

[0028] Autoimmune alopecia

[0029] Autoimmune chronic active hepatitis

[0030] Autoimmune hemolytic anemia

[0031] Autoimmune neutropenia

[0032] Autoimmune thrombocytopenic purpura

[0033] Autoimmune vasculitides

[0034] Behcet's disease

[0035] Cerebellar degeneration

[0036] Chronic active hepatitis

[0037] Chronic inflammatory demyelinating polyradiculoneuropathy

[0038] Dermatitis herpetiformis

[0039] Diabetes

[0040] Eaton-Lambert myasthenic syndrome

[0041] Encephalomyelitis

[0042] Epidermolysis bullosa

[0043] Erythema nodosa

[0044] Gluten-sensitive enteropathy

[0045] Goodpasture's syndrome

[0046] Graft versus host disease

[0047] Guillain-Barre syndrome

[0048] Hashimoto's thyroiditis

[0049] Hyperthyrodism

[0050] Idiopathic hemachromatosis

[0051] Idiopathic membranous glomerulonephritis

[0052] Minimal change renal disease

[0053] Mixed connective tissue disease

[0054] Multifocal motor neuropathy

[0055] Multiple sclerosis

[0056] Myasthenia gravis

[0057] Opsoclonus-myoclonus syndrome

[0058] Pemphigoid

[0059] Pemphigus

[0060] Pernicious anemia

[0061] Polyarteritis nodosa

[0062] Polymyositis/dermatomyositis

[0063] Post-infective arthritides

[0064] Primary biliary sclerosis

[0065] Psoriasis

[0066] Reactive arthritides

[0067] Reiter's disease

[0068] Retinopathy

[0069] Rheumatoid arthritis

[0070] Sclerosing cholangitis

[0071] Sjögren's syndrome

[0072] Stiff-man syndrome

[0073] Subacute thyroiditis

[0074] Systemic lupus erythematosis

[0075] Systemic sclerosis (scleroderma)

[0076] Temporal arteritis

[0077] Thromboangiitis obliterans

[0078] Transplantation rejection

[0079] Type I and type II autoimmune polyglandular syndrome

[0080] Ulcerative colitis

[0081] Uveitis

[0082] Wegener's granulomatosis

[0083] As used herein the term “alkylene” refers to a straight orbranched alkyl chain having two unsatisfied valencies, for example—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂—, —CH(CH₂CH₃)CH₂CH₂CH₃, and—C(CH₃)₃.

[0084] As used herein the term “heteroaryl” refers to a 5- or 6-memberedaromatic ring containing one or more heteroatoms. Illustrative of suchgroups are thienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl,thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl.

[0085] As used herein the unqualified term “heterocyclyl” or“heterocyclic” includes “heteroaryl” as defined above, and in particularmeans a 5-8 membered aromatic or non-aromatic heterocyclic ringcontaining one or more heteroatoms selected from S, N and O, includingfor example, pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl,pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl,morpholinyl, benzofuranyl, pyranyl, isoxazolyl, quinuclidinyl,aza-bicyclo[3.2.1]octanyl, benzimidazolyl, methylenedioxyphenyl,maleimido and succinimido groups.

[0086] Unless otherwise specified in the context in which it occurs, theterm “substituted” as applied to any moiety herein means substitutedwith one or more of the following substituents, namely (C₁-C₆)alkyl,trifluoromethyl, (C₁-C₆)alkoxy (including the special case where a ringis substituted on adjacent ring C atoms by methylenedioxy orethylenedioxy), trifluoromethoxy, (C₁-C₆)alkylthio, phenyl, benzyl,phenoxy, (C₃-C₈)cycloalkyl, hydroxy, mercapto, amino, fluoro, chloro,bromo, cyano, nitro, oxo, —COOH, —SO₂OH, —CONH₂, —SO₂NH₂, —COR^(A),—COOR^(A), —SO₂OR^(A), —NHCOR^(A), —NHSO₂R^(A), —CONHR^(A), —SO₂NHR^(A),—NHR^(A), —NR^(A)R^(B), —CONR^(A)R^(B) or —SO₂NR^(A)R^(B) wherein R^(A)and R^(B) are independently a (C₁-C₆)alkyl group. In the case where“substituted” means substituted by (C₃-C₈)cycloalkyl, phenyl, benzyl orphenoxy, the ring thereof may itself be substituted with any of theforegoing, except (C₃-C₈)cycloalkyl phenyl, benzyl or phenoxy.

[0087] As used herein the unqualified term “carbocyclyl” or“carbocyclic” refers to a 5-8 membered ring whose ring atoms are allcarbon.

[0088] Some compounds of the invention contain one or more chiralcentres because of the presence of asymmetric carbon atoms. The presenceof asymmetric carbon atoms gives rise to stereoisomers ordiastereoisomers with R or S stereochemistry at each chiral centre. Theinvention includes all such stereoisomers and diastereoisomers andmixtures thereof.

[0089] Salts of salt forming compounds of the invention includephysiologically acceptable acid addition salts for examplehydrochlorides, hydrobromides, sulphates, methane sulphonates,p-toluenesulphonates, phosphates, acetates, citrates, succinates,lactates, tartrates, fumarates and maleates; and base addition salts,for example sodium, potassium, magnesium, and calcium salts. Where thecompound contains an amino group, quaternary amino salts are alsofeasable, and are included in the invention.

[0090] In the compounds of the invention the following are examples ofthe several structural variables:

[0091] R₁ may be, for example, H, F, Cl, methyl, methoxy, ormethylenedioxy. Currently it is preferred that R₁ is H, Cl or especiallyF;

[0092] R₂ may be, for example H, methyl, methoxy, cyclopropyl, phenyl,or fluoro-, chloro-, methyl, or methoxy-substituted phenyl. H orcyclopropyl is presently preferred;

[0093] R₃ may be, for example, H, F, Cl, methyl, methoxy, ormethylenedioxy. Currently it is preferred that R₃ is F or Cl, and it ismost preferred that R₃ be H;

[0094] Y may be, for example, —O—, —S—, or —N(R₅)— wherein R₅ representsH or methyl. —NH— or —S— is presently preferred.

[0095] X may be, for example a bond, or a —CH₂— or —CH₂CH₂— radical. Abond is presently preferred.

[0096] R₄ represents —C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆,—NHC(═O)NHR₆, or —NHC(═S)NHR₆. Of these —NR₇C(═O)R₆, and especially—C(═O)NR₆R₇ and —NHC(═O)NHR₆ are curently preferred. R₇ is preferably H,but a wide range of R₆ substituents have given rise to highly activecompounds of the invention. Many exemplary R₆ substituents appear in thecompounds of the Examples below.

[0097] R₆ may be, for example, H or a radical of formula -Alk_(b)-Qwherein b is 0 or 1 and

[0098] Alk may be, for example a —(CH₂)_(n)—,—CH((CH₂)_(m)CH₃)(CH₂)_(n)—, —C((CH₂)_(m)CH₃)((CH₂)_(p)CH₃) (CH₂)_(n)—,—(CH₂)_(n)—O—(CH₂)_(m)—, —(CH₂)_(n)—NH—(CH₂)_(m)—, or—(CH₂)_(n)—NH—(CH₂)_(m)—NH—(CH₂)_(p)— radical where n is 1, 2, 3 or 4and m and p are independently 0, 1, 2, 3 or 4, and

[0099] Q may represent H, —OH, —COOCH₃, phenyl, cyclopropyl,cyclopentyl, cyclohexyl, pyridyl, furyl, thienyl, or oxazolyl; and

[0100] R₇ may be, for example, H, or when taken together with the atomor atoms to which they are attached R₆ and R₇ may form a heterocyclicring of 5, 6 or 7 members.

[0101] Specific examples of R₄ groups include those present in thecompounds of the Examples herein.

[0102] Compounds of the invention may be prepared by synthetic methodsknown in the literature, from compounds which are commercially availableor are accessible from commercially available compounds. For example,compounds of formula (I) wherein R₄ is a group —NR₇C(═O)R₆ may beprepared by acylation of an amine of formula (II) with an acid chlorideof formula (III):

[0103] Compounds of the invention wherein R₄ is a group —NHC(═O)NHR₆ maybe prepared by reaction of an amine of formula (IIA) with an isocyanateof formula (IIIA)

[0104] Compounds of the invention wherein R₄ is a group —C(═O)NHR₆ maybe prepared by reaction of an acid chloride of formula (IIB) with anamine NHR₆R₇:

[0105] Compounds of the invention wherein R₄ is a group —NR₇C(═O)OR₆ maybe prepared by reaction of an amine of formula (II) with a chloroformateClC(═O)OR₆.

[0106] The following Examples illustrate the preparation of compounds ofthe invention:

Preparation of Intermediate 12-(4-Nitrophenyl)-6-fluoro-2,5-dihydropyrazolo[4,3-c]-quinolin-3-one

[0107]

[0108] 4-Nitrophenylhydrazine (2.28 g, 0.014 mol) was added in oneportion to a stirred solution of4-chloro-8-fluoro-quinoline-3-carboxylic acid ethyl ester (3.58 g, 0.014mol) in anhydrous n-butyl alcohol (50 ml) at room temperature. Themixture was refluxed for 16 h under nitrogen, cooled to room temperatureand then filtered to leave an orange solid. The solid was purified bywashing sequentially with ethyl acetate (20 ml) and heptane (20 ml) andthen finally dried under suction to give the pyrazolone (3.93 g, 87%) asa dark orange solid, LCMS m/z 325.24 [M+H]⁺ @ R_(T) 1.47 min.

Preparation of Intermediate 22-(4-Aminophenyl)-6-fluoro-2,5-dihydropyrazolo[4,3-c]-quinolin-3-one

[0109]

[0110] Tin (II) chloride dihydrate (12.5 g, 0.055 mol) was added in oneportion to a stirred solution of2-(4-nitro-phenyl)-6-fluoro-2,5-dihydro-pyrazolo[4,3-c]quinolin-3-one(intermediate 1) (3.59 g, 0.011 mol) in ethyl alcohol (110 ml) at roomtemperature. The mixture was then heated to 80° C. for 8 h, cooled toroom temperature and filtered to leave a yellow solid. The solid wassuspended in a bi-phasic solution of ethyl acetate (1L), a saturatedsolution of Rochelles salt (500 ml) and a saturated solution of sodiumbicarbonate (500 ml) and stirred at room temperature for 2 h. Themixture was filtered and the remaining solid was washed with water anddried under vacuum to afford the title compound (3.39 g, 99%) as abright yellow solid, LCMS m/z 295.30 [M+H]⁺ @ R_(T) 0.84 min.

EXAMPLE 1N-[4-(6-Fluoro-3-oxo-3,5-dihydropyrazolo[4,3-c]quinolin-2-yl)-phenyl]-2-methyl-butyramide

[0111]

[0112] (±)-2-Methylbutyryl chloride (13.6 μl, 0.11 mmol) was addeddropwise over 30 sec to a stirred solution of2-(4-amino-phenyl)-6-fluoro-2,5-dihydro-pyrazolo[4,3-c]quinolin-3-one(Intermediate 2) (30 mg, 0.10 mmol), triethylamine (14 μl, 0.11 mmol)and 4-dimethylaminopyridine (2.4 mg, 0.02 mmol) in dichloromethane (1ml) at room temperature. The mixture was stirred at room temperature for16 h. The yellow solid was then filtered and purified by washingsequentially with a saturated solution of sodium bicarbonate (1 ml),ethyl acetate (1 ml) and ethyl alcohol (0.5 ml) and finally dried undersuction to give the title compound (10 mg, 26%) as a bright yellowsolid, LCMS m/z 379.36 [M+H]⁺ @ R_(T) 1.18 min. δ_(H)(400 MHz, (CD₃)₂SO)9.89 (1H, s), 8.52 (1H, s), 8.15 (2H, d J 9.0 Hz), 8.01 (1H, d J 7.0Hz), 7.69 (2H, d J 9.0 Hz) 7.57-7.46 (2H, m), 2.46-2.39 (1H, m),1.69-1.36 (2H, m), 1.11 (3H, d J 6.8 Hz), 0.91(3H, t J 7.3 Hz).

[0113] The title compound, and compounds of subsequent Examples, weretested in the assay described below in the Assay Section, to determinetheir activities as inhibitors of the CD80-CD28 interaction. The presenttitle compound had an activity rating of ***.

EXAMPLES 2-49

[0114] The following compounds were synthesized by the route describedin Example 1, substituting the appropriate acid chloride for(±)-2-methylbutyryl chloride:

EXAMPLE 2 2-Methyl-pentanoic acid[4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-amide

[0115]

[0116] δ_(H)(400 MHz, (CD₃)₂SO) 9.92 (1H, s), 8.53 (1H, s) 8.12 (2H, d J9.2 Hz), 8.05 (1H, d J 7.6 Hz), 7.70 (2H, d J 9.2 Hz), 7.63-7.53 2H, m),1.68-1.58 (1H, m), 1.38-1.28 (3H, m), 1.11 (3H, d J 6.6 Hz), 0.91 (3H, tJ 7.1 Hz).

[0117] Activity ***

EXAMPLE 3 1-Methyl-1H-pyrrole-2-carboxylic acid[4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-amide

[0118]

[0119] δ_(H)(400 MHz, (CD₃)₂SO) 9.76 (1H, s), 8.50 (1H, s), 8.26 (2H, d9.0 Hz), 7.97-7.94 (1H, m), 7.73 (2H, d J 9.0 Hz), 7.39-7.28 (2H, m),7.07-7.01 (2H, m), 3.91 (3H, s)

[0120] Activity *

EXAMPLE 4N-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-3-methyl-butyramide

[0121]

[0122] δ_(H)(400 MHz, (CD₃)₂SO) 9.92 (1H, s), 8.52 (1H, s), 8.14 (2H, dJ 9.2 Hz), 8.01 (1H, d J 7.3 Hz), 7.67 (2H, d J 9.2 Hz), 7.57-7.47 (2H,m), 2.21 (2H, d J 6.8 Hz), 2.14-2.07 (1H, m), 0.96 (6H, d J 6.6 Hz).

[0123] Activity **

EXAMPLE 5 2-Propyl-pentanoic acid[4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-amide

[0124]

[0125] δ_(H)(400 MHz, (CD₃)₂SO) 9.93 (1H, s), 8.53 (1H, s), 8.11 (2H, dJ 9.0 Hz), 8.05 (1H, d J 7.8 Hz), 7.70 (2H, d J 9.0 Hz), 7.59-7.46 (2H,m), 2.46-2.35 (1H, m), 1.63-1.27 (4H, m), 0.90(6H, t J 7.1 Hz).

[0126] Activity *

EXAMPLE 65-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)phenylcarbamoyl]-pentanoicacid methyl ester

[0127]

[0128] δ_(H)(400 MHz, (CD₃)₂SO) 9.85 (1H, s), 8.47 (1H, s), 8.25 (2H, dJ 9.0 Hz), 7.91-7.90 (1H, m), 7.59 (2H, d J 9.0 Hz), 7.29-7.20 (2H, m),3.61 (3H, s), 2.38-2.28 (4H, m), 1.64-1.50 (4H, m)

[0129] Activity ***

EXAMPLE 7N-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-2,2-dimethyl-propionamide

[0130]

[0131] δ_(H)(400 MHz, (CD₃)₂SO) 9.26 (1H, S), 8.52 (1H, s), 8.15 (2H, dJ 9.2 Hz), 8.03 (1H, d J 8.8 Hz), 7.71 (2H, d J 9.2 Hz), 7.56-7.47 (2H,m), 1.26 (9H, s)

[0132] Activity **

[0133] Examples 8 to 28 were also prepared by the method of Example 1using the appropriate acid chloride:

M.S. Example X R (MH+) Activity 8 6-F

443.4 ** 9 6-F —CH₂Cl 371.31 ** 10 6-F

389.34 * 11 6-F

485.45 * 12 6-F CO₂Me 381.34 ** 13 6-F OEt 367.18 14 6-F

507.43 * 15 6-F

466.41 ** 16 6-F Me 337.36 ** 17 6-F CH(Et)CH₂CH₂CH₂Me 421.46 * 18 6-FCH(Et)₂ 393.41 *** 19 6-F

405.41 ** 20 6-F

448.44 ** 21 6-F

481.35 ** 22 6-F

423.42 *** 23 6-F (CH₂)₈CO₂Me 493.51 ** 24 6-F iPr 365.36 *** 25 6-FCH₂OCH₂CH₂OMe 411.4 ** 26 6-F CH(Me) (nPr) 393.42 *** 27 6-F CH₂OMe367.24 ** 28 6-F

390.33 ** 29 6-F CH₂CH₂CH₂N⁺(Me)₃ 422.1 (M+) *** 30 6-F CH₂CH₂CH₂N(Me)₂408.3 *** 31 6-F CH₂NHCH₂CH₂CH₂N(Me) (Ph) 499.3 * 32 6-F

485.3 * 33 6-F

505.1 *** 34 6-F

517.2 *** 35 6-F

477.1 *** 36 6-F

457.1 ** 37 6-F

463.1 ** 38 6-F

438.3 ** 39 6-F

463.2 *** 40 6-F

460.4 ** 41 6-F CH₂NHCH₂CH₂N(iPr)₂ 479.4 ** 42 6-F

420.2 ** 43 H CH(NH₂)CH₃ 348.3 ** 44 H CH(Me)nPr 375.3 * 45 H iPr 347.3** 46 6-F CH(NH₂)CH₃ 366.3 *** 47 H CH(Me)Et 361.3 ** 48 6-F

529.1 ** 49 6-F CH₂N(Me)CH₂Ph 456.4 **

Preparation of Intermediate 33-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid

[0134]

[0135] 3-Hydrazinobenzoic acid (1.91 g, 0.013 mol) was added in oneportion to a stirred solution of4-chloro-8-fluoro-quinoline-3-carboxylic acid ethyl ester (2.93 g, 0.011mol) in n-butanol (60 ml) at room temperature. The solution was heatedto reflux for 16 h, cooled to room temperature and the resulting yellowsolid filtered, washed with tert-butyl methyl ether and then dried. Thesolid was redissolved in a solution of tetrahydrofuran:water (2:1; 21ml) and lithium hydroxide (1.27 g, 0.031 mol) was then added. Afterstirring at room temperature for 16 h, concentrated hydrochloric acid (3ml) was added dropwise to the mixture to precipitate a yellow solidwhich was filtered and dried under vacuum to give the title compound(intermediate 3) (2.32 g, 63%) as a bright yellow solid.

Preparation of Intermediate 43-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylchloride

[0136]

[0137] Oxalyl chloride (20 ml, 0.2 mol) was added dropwise over 2 min toa stirred solution of3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid(intermediate 3) (2.0 g, 6.1 mmol) in dichloromethane (10 ml) at roomtemperature. N,N-Dimethylformamide (50 μl) was then added and theresulting mixture heated to 50° C. for 1 h. The solution was then cooledto room temperature and then concentrated in vacuo to leave the titlecompound (intermediate 4) (2.0 g, 96%) as a beige solid.

EXAMPLE 503-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-N-(3-methoxy-propyl)-benzamide

[0138]

[0139] 3-Methoxypropylamine (0.026 g, 0.29 mmol) was added to a stirredsolution of3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylchloride (intermediate 4) (26 mg 0.29 mmol) in tetrahydrofuran (2 ml)and the mixture stirred at room temperature for 15 min. Triethylamine(0.2 ml, 1.4 mmol) was then added and the resulting mixture stirredovernight. 1 M Hydrochloric acid (3-4 ml) was added dropwise toprecipitate a yellow solid which was filtered and dried under suction togive the amide (79 mg, 0.20 mmol) as a yellow solid, LCMS m/z 395.25[M+H]⁺ @ R_(T) 1.04 min; δ_(H)(400 MHz, (CD₃)₂SO) 8.59 (1H, m), 8.57(1H, s), 8.39 (1H, app d J 9.3 Hz), 8.08 (1H, app d J 7.3 Hz), 7.66-7.53(5H, m), 3.37-3.33 (4H, m), 3.27 (3H, s), 1.83-1.77 (2H, m).

[0140] Activity **

EXAMPLE 51N-Ethyl-3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl)-benzamide

[0141]

[0142] Prepared by the method of Example 53 substituting ethylamine for3-methoxypropylamine.

[0143] δ_(H)(400 MHz, (CD₃)₂SO) major rotomer quoted; 8.56 (1H, br s),8.47 (1H, m), 8.21 (2H, d J 8.5 Hz), 7.94 (2H, d J 8.5 Hz), 3.96 (3H,s), 3.31 (2H, q J 7.3 Hz), 2.58 (3H, s), 1.15 (3H, t J 7.4 Hz).

[0144] Activity **

EXAMPLE 52N-Benzyl-3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl)-benzamide

[0145]

[0146] Prepared by the method of Example 53 substituting benzylamine for3-methoxypropylamine.

[0147] LCMS m/z 427.16 [M+H]⁺ @ R_(T) 1.28 min.

[0148] Activity *

[0149] Examples 53 to 64 were prepared by the method of example 50,using the appropriate amine.

M.S. Example X R R′ (MH+) Activity 53 6-F CH₂CH₂CH₂N(Me)₂ Me 422.5 * 546-F CH₂CH_(2l CH) ₂N(Me)₂ H 408.4 ** 55 6-F

H 420.4 * 56 6-F

H 434.4 * 57 6-F

H 448.4 ** 58 6-F CH₂CH₂CH₂CH₂N(Me)₂ H 422.4 ** 59 6-F CH₂CH₂OMe H 381.3** 60 6-F Et Et 379.3 * 61 6-F CH₂CO₂Me H 395.2 * 62 6-F CH₂CCH H 361.3** 63 6-F CH₂Ph Me 427.2 ** 64 6-F

463.3 *

EXAMPLE 65 N-(3-Dimethylaminopropyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c)quinolin-2-yl]-benzamideStep 1 2-cyclopropyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethylester

[0150]

[0151] A solution of 3-cyclopropyl-3-oxo-propionic acid methyl ester(6.2 g, 0.038 mols), 2-amino benzoic acid ethyl ester (4.95 g, 0.03mols) and p-toluene sulfonic acid (0.04 g, 0.2 mmols) in toluene (25 ml)was heated at 125° C. for 2 h; 15 ml of solvent was then distilled. Tothe residual orange solution was added sodium ethoxide (2 M, 15 ml) inethanol (reaction mixture turns red). This red mixture was stirred at120° C. for 2 h; 15 ml of solvent was again distilled. The reactionmixture was left to cool to room temperature, diluted with ethyl acetate(1 litre), extracted with HCl 0.1 M and water. The combined organicextracts were dried over sodium sulfate and concentrated in vacuo toleave an orange residue which was washed once with cold ethyl acetate toyield 2-cyclo-propyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethylester (3.87 g, 53%) as an off-white solid. LCMS m/z 244.14 [M+H]⁺ @R_(T) 0.78 min, 89%, m/z 230.11 [Acid+H]⁺ @ R_(T) 1.27, 11%.

[0152] δ_(H)(400 MHz, (CD₃)₂SO) 11.04 (1 H, s), 8.06 (1 H, dd, J₁ 1.1,J₂ 8.1), 7.76-7.66 (2 H, m), 7.36 (1 H, td, J₁ 1.1, J₂ 7.5), 3.89 (3 H,s), 2.16 (1 H, m), 1.18 (4 H, d, J 7.0).

Step 2 4-Chloro-2-cyclopropyl-quinoline-3-carboxylic acid ethyl ester

[0153]

[0154] Phosphorus oxychloride (0.77 ml, 0.082 mols) was added in oneportion to a suspension of2-cyclopropyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethyl ester(1.0 g, 0.041 mols) in acetonitrile and the mixture was heated at 75° C.for 90 minutes (becomes a clear solution above 65° C.). The resultinglight brown solution was poured into saturated sodium bicarbonate (100ml); the suspension was extracted with ethyl acetate and the combinedorganic extracts were dried and concentrated in vacuo to leave4-Chloro-2-cyclopropyl-quinoline-3-carboxylic acid ethyl ester (1.15 g,106%) as an off-white solid. R_(f)(AcOEt)=0.73.

Step 34-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid

[0155]

[0156] 4-Chloro-2-cyclopropyl-quinoline-3-carboxylic acid ethyl ester(1.15 g, 0.0041 mols) and 4-hydrazino-benzoic acid (1.0 g, 0.0068 mols)were stirred in ethanol (30 ml) at reflux for 16 h. The bright yellowsuspension was diluted with heptane, filtered, washed with coldt-butylmethyl ether and left to dry under suction to yield crude solidcontaining hydrazine. This solid was suspended in 1 M HCl, filtered,washed with water and then dried in vacuo to yield4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid (1.135 g, 80%) as a yellow solid, LCMS m/z 346.20 [M+H]⁺ @ R_(T)1.05 min: 96% purity.

[0157] δ_(H)(400 MHz, (CD₃)₂SO) 11.4 (1 H, s), 8.43 (2 H, d, J 8.1),8.21 (1 H, dd, J₁ 1.2, J₂ 8.1), 8.07 (2 H, d, J 8.1), 7.92 (1 H, d, J8.1), 7.67 (1 H, t, J 6.6), 7.52 (1 H, t, J 6.5), 3.43 (1 H, m), 1.59 (2H, m), 1.43 (2 H, m).

Step 44-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl)-benzoylchloride

[0158]

[0159] To a suspension of finely ground4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid (0.19 g. 0.55 mmol) in dichloromethane (4 ml) was added oxalylchloride (1.6 ml, 0.01 mol) followed by a drop of dimethyl formamide.The mixture was stirred under nitrogen at 45° C. for 8 h. The solventwas removed in vacuo to yield4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylchloride as a pale yellow solid, LCMS m/z [M+MeOH—Cl]⁺ @ R_(T) 1.46 min:95% purity. Used without further purification.

Step 5 N-(3-Dimethylaminopropyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzamide

[0160]

[0161] To a partial solution of4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylchloride (0.1 g, 0.28 mmol) in tetrahydrofurane (6 ml) under nitrogenwas added a solution of 3-dimethylamino-propyl amine (0.03 g, 0.3 mmol)in tetrahydrofurane (3 ml). The mixture was stirred at R_(T) for 3 h.The solvent was removed under reduced pressure and the yellow solid waswashed with a little saturated sodium bicarbonate, water and dried undervacuo to yield N-(3-Dimethylaminopropyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl]-benzamide(57 mg, 47%) as a yellow solid. LCMS m/z 430.11 [M+H]⁺ @ R_(T) 0.99 min:100% purity.

[0162] Activity ***

Preparation of Intermediate 54-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzoylchloride

[0163]

[0164] To a suspension of finely ground4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzoic acid(1.1 g. 3.4 mmol) in dichloromethane (6 ml) was added oxalyl chloride(2.4 ml, 29 mmol) followed by a drop of dimethyl formamide. The mixturewas stirred under nitrogen at 45° C. for 3 h. The solvent was removed invacuum to yield 4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzoyl chloride (1.15 g, quantitative) as a paleyellow solid that was used without further purification.

EXAMPLE 66 N-(3-Dimethylaminopropyl)-4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzamidehydrochloride

[0165]

[0166] To a partial solution of4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzoylchloride (0.1 g, 0.3 mmol) in tetrahydrofurane (5 ml) under nitrogen wasadded a solution of 3-dimethylamino-propyl amine (0.03 g, 0.3 mmol) intetrahydrofurane. The mixture was stirred at rt for 90 minutes. Thesolvent was removed under reduced pressure and the yellow solid waspurified via FCC silica gel (gradient elution, MeOH:H₂O, Fluka C₁₈reverse phase) to yield N-(3-Dimethylaminopropyl)-4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzamidehydrochloride (70 mg, 53%) as a yellow solid.

[0167] LCMS m/z 408.39 [M+H]⁺ @ R_(T) 0.89 min: 90% purity.

[0168] Activity ***

EXMAPLES 67-141 Were Prepared Analogously From the Appropriate BenzoylChloride and the Appropriate Amine

[0169]

M.S. Example X Z W R R′ (MH+) Activity 67 6-F H H —CH₂CH₂CH₂CH₂CH₂—391.3 ** 68 6-F H H —CH₂Phenyl H 413.2 *** 69 6-F H H —CH₂Phenyl Me427.3 ** 70 6-F H H —CH₂CH₂OMe H 381.2 *** 71 6-F H H —CH₂CH₂N(Me)₂ H394.3 *** 72 6-F H H —CH₂CO₂Me H 395.3 *** 73 6-F H H —CH₂CH₂CH₂OMe H395.2 *** 74 6-F H H —CH₂CH₂CH₂N(Me)₂ H 408.3 *** 75 6-F H H

H 431.3 ** 76 6-F H H

H 419.2 ** 77 6-F H H Et H 351.2 *** 78 6-F H H Et Et 379.3 ** 79 6-F HH

H 420.4 *** 80 6-F H H —CH₂CH₂CH₂N(Me)₂ Me 422.4 *** 81 6-F H H—CH₂CH₂CH₂CH₂N(Me)₂ H 422.4 *** 82 6-F H H

H 448.5 *** 83 6-F H H

H 434.4 *** 84 6-F H H

H 525.3 *** 85 6-F H H —CH₂CH₂CH₂CH₂CH₂N(Me)₂ H 450.3 *** 86 H H H—CH₂CH₂CH₂N(Me)₂ H 390.2 *** 87 H H H —CH₂CH₂CH₂CH₂CH₂N(Me)₂ H 432.1 **88 H H H —CH₂CH₂CH₂CH₂N(Et)₂ H 432.2 ** 89 H H H —CH₂CH₂CH₂N(Me)₂ Me404.2 ** 90 6-F H 2—Cl —CH₂CH₂CH₂N(Me)₂ H 442.1 ** 91 H H H

H 416.1 ** 92 H H H

H 573.0 ** 93 H H H

H 445.1 ** 94 H H H

H 507.1 ** 95 6-F H H

H 591.0 *** 96 H

H —CH₂CH₂CH₂N(Me)₂ H 430.1 *** 97 6-F H H

H 464.1 *** 98 6-F H H

H 463.1 *** 99 6-F H 3—Cl

H 482.1 ** 100 6-F H 2—Cl

H 497.1 ** 102 6-F H 2—Cl —CH₂CH₂CH₂CH₂N(Et)₂ H 484.1 ** 103 6-F H 3—Cl—CH₂CH₂CH₂N(Me)₂ H 442.1 ** 104 H

H

H 470.4 *** 105 6-F H H

516.3 * 106 6-F H H

H 470.3 *** 107 6-F H H —CH₂CH₂N(iPr)₂ H 451.4 *** 108 6-F H 2—Cl

H 496.2 ** 109 6-F H H

H 456.1 *** 110 6-F H 2—Cl —CH₂CH₂CH₂CH₂N(Me)₂ H 456.1 ** 111 6-F H H

406.2 ** 112 6-F H H

H 462.1 *** 113 6-F H H

H 436.1 *** 114 6-F H H

H 434.4 *** 115 6-F H H

H 476.1 *** 116 6-F H H

H 496.1 *** 117 6-F H H

H 436.3 *** 118 6-F H H

H 462.3 *** 119 6-F H H

H 428.1 ** 120 6-F H H —CH₂CH₂SEt H 411.3 *** 121 6-F H H

H 448.3 ** 122 6-F H H

H 431.3 *** 123 6-F H H

H 434.3 ** 124 6-F H H —CH₂CH₂CH₂CH₂N(Et)₂ H 450.4 *** 125 6-F

H

H 536.1 *** 126 6-F

H

H 516.2 *** 127 6-F H H

H 428.3 * 128 6-F H H —CH₂CH₂CH₂SMe H 411.3 ** 129 H

H

H 498.5 *** 130 6-F

H

H 488.4 *** 131 6-F H H

H 446.3 *** 132 6-F

H —CH₂CH₂CH₂N(Me)₂ H 448.2 *** 133 6-F

H

H 502.3 *** 134 6-F

H

H 486.3 *** 135 6-F

H —CH₂CH₂CH₂CH₂N(Et)₂ H 490.3 *** 136 6-F

H

H 546.2 ** 137 6-F

H

H 631.2 *** 138 6-F

H

H 468.2 ** 139 6-F

H

H 468.2 * 140 6-F

H

H 476.2 *** 141 6-F

H

H 474.3 ***

EXAMPLE 142{3-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-ureido}aceticacid ethyl ester

[0170]

[0171] Ethyl cyanatoacetate (31 mg, 0.24 mmol) was added in one portionto a stirred solution of2-(4-aminophenyl)-6-fluoro-2,5-dihydropyrazolo[4,3-c]quinolin-3-one(intermediate 2) (50 mg, 0.17 mmol) in N,N-dimethylformamide (2 ml) andthe mixture stirred at room temperature for 16 h. Water (1 ml) was thenadded to the mixture to precipitate a solid, which was filtered, washedwith water (1 ml) and then ethyl acetate (1 ml) and finally dried bysuction to leave the urea as a yellow solid, LCMS m/z 424.40 [M+H]⁺ @R_(T) 1.06 min.

[0172] Activity ***

EXAMPLES 143 and 144

[0173]

Example 143 LCMS m/z 438.41 [M + H^(]+)@ RT 1.13 min. Activity **

Example 144 LCMS m/z 514.46 [M + H^(]+)@ RT 1.35 min. Activity *

[0174] The following compounds were synthesised by the method of Example142, substituting the appropriate isocyanate, isothiocyanate orchloroformate for ethyl cyanatoacetate.

M.S. Example X Z Y R A (MH +) Activity 144 6-F H O iPr NH 380.3 *** 1456-F H O nPr NH 380.3 *** 146 6-F H O tBu NH 394.4 *** 147 6-F H O Ph NH414.3 ** 148 6-F H S

NH 394.3 ** 149 6-F H S

NH 436.4 * 150 6-F H O tBu O 395.3 *** 151 6-F H O Et O 367.2 ** 152 6-FH O CH₂CH₂N(Me)₂ O 410.2 *** 153 H

O Me O 375.3 ** 154 6-F H O CH₂CH₂CH₂N(Me)₂ O 424.1 *** 155 6-F H O

O 512.3 ** 156 6-F H S nPentyl NH 424.4 ** 157 6-F H S CH(CH₃)CH(CH₃)CH₃NH 424.4 ** 158 6-F H O CH₂CH₂CH₂CH₂N(Et)₂ NH 465.4 *** 159 H H O nPr NH362.3 *** 160 H H S

NH 376.1 ** 161 6-F H O CH₂CH₂CH₂N(Me)₂ NH 423.3 *** 162 H H O

NH 434.5 *** 163 6-F H O CH₂CH₂CH₂CH₂N(Me)₂ NH 437.2 *** 164 6-F H O

NH 463.5 ***

Intermediate 6: Preparation of methyl 4-oxothiochromane-3-carboxylate

[0175]

[0176] Dry tetrahydrofuran (60 ml) was cooled under nitrogen atmosphereto −50 to −60° C. 1M Lithium bis(trimethylsily)amide solution in hexane(56 ml, 56 mmol) was added. The temperature was kept at −50 to −60° C.and thiochroman-4-one was added dropwise over 20 min. Stirring wascontinued at low temperature for 60 min. Methyl cyanoformate (4.84 ml,60.9 mmol) was added dropwise over 5 min to the reaction mixture. Theobtained suspension was stirred at −50 to −60° C. for 80 min and thenallowed to warm up to room temperature. Saturated ammonium chloridesolution (100 ml) was added. The phases were separated, the aqueousphase extracted with ethyl acetate (2×100 ml). The combined organicphases were washed with water (50 ml), dried over magnesium sulphate,filtered and concentrated under vacuum. An orange oil was obtained andpurified by column chromatography. The title compound was isolated as ayellow solid (4.70 g, 21.1 mmol, 42%). LCMS: m/z 221 [M−H]⁺.

Intermediate 7: Preparation of4-(3-Oxo-3a,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzoic acid

[0177]

[0178] 4-Oxothiochromane-3-carboxylate (0.50 g, 2.25 mmol) andhydrazinobenzoic acid (0.377 g, 2.48 mmol) were mixed in acetic acid (6ml). The mixture was heated to reflux for 30 min. Excess acetic acid wasdistilled off to give a brown oil. Diethylether was added, a precipitateformed which was collected by filtration and dried under vacuum. Thecrude product was isolated as a red/brown solid (797 mg). LCMS: m/z 325[M+H]⁺. No purification was carried out.

Intermediate 8: Preparation of4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzoic acid

[0179]

[0180] Crude4-(3-Oxo-3a,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzoic acid(250 mg, 0.77 mmol) was dissolved in dimethyl sulphoxide (6 ml).O-Chloranil (189 mg, 0.77 mmol) was added and the mixture was stirred atroom temperature overnight. Water (20 ml) was added and the solids werecollected by filtration and washed with water. The filter cake wastriturated with toluene, filtered and dried under vacuum. The titlecompound was isolated as a dark brown solid (230 mg, 0.71 mmol, 92%).LCMS: m/z 323 [M+H]⁺

[0181] Alternatively crude4-(3-Oxo-3a,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzoic acidcan be stirred in dimethyl sulphoxide under exposure to air. It wasfound that air oxidation provides clean product, however the reaction ismuch slower.

EXAMPLE 165 Preparation ofN-[3-(dimethylamino)propyl]-4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzamide

[0182]

[0183] 4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzoic acid (55 mg,0.17 mmol) was suspended in anhydrous dimethyl acetamide (1 ml).Diisopropyl-ethyl amine (46.5 mg, 0.36 mmol, 62μl) was added followed by3-dimethylaminopropylamine (17.5 mg, 0.17 mmol) and[(benzotriazol-1-yloxy)-dimethylamino-methylene]-dimethyl-ammoniumhexafluoro phosphate (65 mg, 0.17 mmol). The mixture was stirred at roomtemperature for 4 h and was purified by preparative HPLC. The titlecompound was isolated as a brown solid. LCMS: m/z 407 [M+H]⁺

[0184] Activity **

EXAMPLE 166 Preparation ofN-[(cyclohexylamino)propyl]-4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzamide

[0185]

[0186] The reaction was carried out as described above. LCMS: m/z 461[M+H]⁺

[0187] Activity ***

EXAMPLE 167 Preparation ofN-(pyrrolidin-1-yl-butyl)-4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzamide

[0188]

[0189] The reaction was carried out as described above. LCMS: m/z 447[M+H]⁺

[0190] Activity *

EXAMPLE 168 Preparation of4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)-N-1,2,2,6,6-pentamethylpiperidin-4-ylbenzamide

[0191]

[0192] The reaction was carried out as described above. LCMS: m/z 475[M+H]⁺

[0193] Activity **

Intermediate 9: Preparation of 3-[(2-fluorophenyl)sulfanyl]propanoicacid

[0194]

[0195] 2-Fluorothiophenol (5.0 g, 39 mmol) was dissolved intetrahydrofuran (50 ml) under a nitrogen atmosphere. Triethylamine (3.94g, 5.33 ml, 85.8 mmol) was added. Acrylic acid (2.81 g, 2.67 ml, 39mmol) was dissolved in tetrahydrofuran and added dropwise to thereaction solution over 2 h at room temperature. The mixture was stirredat room temperature overnight. 1M Hydrochloric acid (50 ml) was addedand the phases were separated. The aqueous phase was washed with ethylacetate (2×50 ml). The combined organic phases were dried over magnesiumsulphate, filtered and concentrated under vacuum. A yellow oil wasobtained which solidified upon storage at room temperature. The solidwas triturated with hexane, filtered and dried under vacuum. The titlecompound was isolated as an off-white solid (4.19 g, 20.9 mmol, 54%).

Intermediate 10: Preparation of8-fluoro-2,3-dihydro-4H-thiochromen-4-one

[0196]

[0197] 3-[(2-Fluorophenyl)sulfanyl]propanoic acid (4.0 g, 20 mmol) wasmixed with concentrated sulphuric acid (20 ml) at 0-5° C. The reactionsolution was stirred at 0 to 5° C. for 3 h then allowed to warm up toroom temperature overnight. The mixture was quenched dropwise into iceto give a white suspension. The aqueous phase was extracted with ethylacetate (1×200 ml, 1×100 ml). The combined organic phases were washedwith saturated sodium bicarbonate solution (1×50 ml), water (1×50 ml),1M hydrochloric acid (50 ml) and water (2×50 ml). The organic phase wasdried over magnesium sulphate, filtered and concentrated under vacuum.The title compound was isolated as a yellow solid (2.10 g, 11.5 mmol,58%).

Intermediate 11: Preparation of methyl8-fluoro-4-oxothiochromane-3-carboxylate

[0198]

[0199] 1M Lithium hexamethyldisilazide solution in hexane (13.2 ml) wasdissolved in anhydrous tetrahydrofuran (20 ml) under nitrogenatmosphere. The solution was cooled to −78° C.8-Fluoro-2,3-dihydro-4H-thiochromen-4-one (2.00 g, 11 mmol) wasdissolved in tetrahydrofuran (40 ml), the solution was transferred tothe dropping funnel and added dropwise over 30 min to the reactionmixture maintaining the temperature below −60° C. An orange clearsolution was obtained which was stirred at −78° C. to −65° C. for 2 h.Methyl cyanoformate (0.935 g, 0.87 ml) was dissolved in tetrahydrofuran(2 ml) and added dropwise to the reaction solution. Stirring wascontinued at low temperature for 1 h, the mixture was then allowed towarm to room temperature. Saturated ammonium chloride solution (20 ml)and water (10 ml) were added, the phases mixed for 5 min and separated.The aqueous phase was washed with ethyl acetate (2×100 ml) and thecombined organic phases were dried over magnesium sulphate. The mixturewas filtered and the solvent removed under vacuum to give an orange oil.The crude oil was purified by column chromatography; mobile phase:hexanes, gradient to hexanes/ethyl acetate [90:10]. The title compoundwas isolated as a yellow solid (1.19 g, 4.95 mmol, 45%).

Intermediate 12: Preparation of4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzoic acid

[0200]

[0201] Methyl 8-fluoro-4-oxothiochromane-3-carboxylate (1.19 g, 4.95mmol) and 4-hydrazinobenzoic acid (755 mg, 4.95 mmol) were mixed withglacial acetic acid (10 ml). The mixture was heated to reflux for 4 h.Excess acetic acid was removed under vacuum to give an orange oil. Ethylacetate (10 ml) was added and the mixture sonicated. Precipitation of anorange solid was observed. The solids were collected by filtration andwashed with ethyl acetate. The filter cake was taken up in dimethylsuphoxide (10 ml) and air-oxidised at room temperature for one week.Water (20 ml) was added to the reaction mixture, the solids werecollected by filtration, slurried in ethyl acetate, filtered and driedunder vacuum. The title compound was isolated as an orange powder (175mg, 0.51 mmol, 10%). LCMS: m/z 341.

EXAMPLE 169 Preparation ofN-[3-(dimethylamino)propyl]-4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzamide

[0202]

[0203] 4-(6-Fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzoic acid(41 mg, 0.12 mmol) was dissolved in anhydrous dimethyl-acetamide(1 ml).Diisopropyl-ethyl amine (46 mg, 0.36 mmol, 62 μl) was added followed by[(benzotriazol-1-yloxy)-dimethylamino-methylene]-dimethyl-ammoniumhexafluoro phosphate (65 mg, 0.17 mmol) and 3-dimethylaminopropylamine(12 mg, 0.12 mmol). The mixture was stirred at room temperatureovernight and purified by preparative HPLC. The title compound wasisolated as a brown solid. LCMS: m/z 425 [M+H]^(+.)

[0204] Activity **

EXAMPLE 170 Preparation ofN-[(cyclohexylamino)propyl]-4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzamide

[0205]

[0206] The reaction was carried out as described above. LCMS: m/z 479[M+H]⁺.

[0207] Activity **

EXAMPLE 171 Preparation ofN-(pyrrolidin-1-yl-butyl)-4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzamide

[0208]

[0209] The reaction was carried out as described above. LCMS: m/z 465[M+H]⁺.

[0210] Activity ***

EXAMPLE 173 Preparation of4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)-N-1,2,2,6,6-pentamethylpiperidin-4-ylbenzamide

[0211]

[0212] The reaction was carried out as described above. LCMS: m/z 493[M+H]⁺

[0213] Activity ***

Assay Section

[0214] The examples described above were tested in a cell freeHomogenous Time Resolved Fluorescence (HTRF) assay to determine theiractivity as inhibitors of the CD80-CD28 interaction.

[0215] In the assay, europium and allophycocyanin (APC) are associatedwith CD28 and CD80 indirectly (through antibody linkers) to form acomplex, which brings the europium and APC into close proximity togenerate a signal. The complex comprises the following six proteins:fluorescent label 1, linker antibody 1, CD28 fusion protein, CD80 fusionprotein, linker antibody 2, and fluorescent label 2. The table belowdescribes these reagents in greater detail. Fluorescent Anti-Rabbit IgGlabelled with Europium label 1 (1 μg/ml) Linker Rabbit IgG specific formouse Fc antibody 1 fragment (3 μg/ml) CD28 fusion CD28 - mouse Fcfragment fusion protein protein (0.48 μg/ml) CD80 fusion CD80 mouse Fabfragment (C215) fusion protein protein (1.9 μg/ml) Linker GαMκ-biotin:biotinylated goat IgG antibody 2 specific for mouse kappa chain (2μg/ml) Fluorescent SA-APC: streptavidin labelled label 2 allophycocyanin(8 μg/ml)

[0216] On formation of the complex, europium and APC are brought intoproximity and a signal is generated.

[0217] Non-specific interaction was measured by substituting a mouse Fabfragment (C215) for the CD80 mouse Fab fragment fusion protein (1.9μg/ml). The assay was carried out in black 384 well plates in a finalvolume of 30μl. Assay buffer: 50 mM Tris-HCl, 150 mM NaCl pH 7.8,containing 0.1% BSA (w/v) added just prior to use.

[0218] Compounds were added to the above reagents in a concentrationseries ranging between 100 μM-1.7 nM. The reaction was incubated for 4hours at room temperature. Dual measurements were made using a WallacVictor 1420 Multilabel Counter. First measurement: excitation 340 nm,emission 665 nm, delay 50 μs, window time 200 μs. second measurement:excitation 340 nm, emission 615 nm, delay 50 μs, window time 200 μs.Counts were automatically corrected for fluorescence crossover,quenching and background.

[0219] By way of illustration, the EC₅₀ results for the compounds ofExamples 15, 21, 29, 35 and 83 were 8 μM, 1.9 μM, 950 nM, 148 nM and 90nM respectively. For convenience, the EC50 activities of compoundstested are recorded above in summary form as:

EC50: *=>10 μM, **=1-10 μM, ***=<1 μM.

1. A compound of formula (I) or a pharmaceutically veterinarilyacceptable salt thereof:

wherein R₁ and R₃ independently represent H; F; Cl; Br; −NO₂; —CN; C₁-C₆alkyl optionally substituted by F or Cl; or C₁-C₆ alkoxy optionallysubstituted by F; R₂ represents H, or optionally substituted C₁-C₆alkyl, C₃-C₇ cycloalkyl or optionally substituted phenyl; Y represents—O—, —S—, N-oxide, or —N(R₅)— wherein R₅ represents H or C₁-C₆ alkyl; Xrepresents a bond or a divalent C₁-C₆ alkylene radical; R₄ represents—C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆, —NHC(═O)NHR₆ or —NHC(═S)NHR₆wherein R₆ represents H, or a radical of formula -(Alk)_(b)-Q wherein bis 0 or 1 and Alk is an optionally substituted divalent straight chainor branched C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene or C₂-C₁₂ alkynyleneradical which may be interrupted by one or more non-adjacent —O—, —S— or—N(R₈)— radicals wherein R₈ represents H or C₁-C₄ alkyl, C₃-C₄ alkenyl,C₃-C₄ alkynyl, or C₃-C₆ cycloalkyl, and Q represents H; —CF₃; —OH; —SH;—NR₈R₈ wherein each R₈ may be the same or different; an ester group; oran optionally substituted phenyl, C₃-C₇ cycloalkyl, C₅-C₇ cycloalkenylor heterocyclic ring having from 5 to 8 ring atoms; and R₇ represents Hor C₁-C₆ alkyl; or when taken together with the atom or atoms to whichthey are attached R₆ and R₇ form an optionally substituted heterocyclicring having from 5 to 8 ring atoms.
 2. A compound as claimed in claim 1wherein R₁ is H, F, Cl, methyl or methoxy.
 3. A compound as claimed inclaim 1 or claim 2 wherein R₂ is H, methyl, methoxy, cyclopropyl,phenyl, or fluoro-, chloro-, methyl, or methoxy-substituted phenyl.
 4. Acompound as claimed in any of the preceding claims wherein R₃ is H, F,Cl, methyl, methoxy, or methylenedioxy.
 5. A compound as claimed in anyof the preceding claims wherein Y is —O—, —S—, or —N(R₅)— wherein R₅represents H or methyl.
 6. A compound as claimed in any of the precedingclaims wherein X is a bond, or a —CH₂— or —CH₂CH₂— radical.
 7. Acompound as claimed in any of the preceding claims wherein R₄ represents—C(═O)NHR₆, —NR₇C(═O)R₆, —NR₇C(═O)OR₆, —NHC(═O)NHR₆ or —NHC(═S)NHR₆ andin these R₆ is H or a radical of formula -Alk_(b)-Q wherein b is 0 or 1and Alk is a —(CH₂)_(n)—, —CH((CH₂)_(m)CH₃)(CH₂)_(n)—,—CH((CH₂)_(m)CH₃)((CH₂)_(p)CH₃)(CH₂)_(n)—, —(CH₂)_(n)—O—(CH₂)_(m)—, or—(CH₂)_(n)—)—(CH₂)_(n)O—(CH₂)_(m)—, radical where n is 1, 2, 3 or 4 andm and p are independently 0, 1, 2, 3 or 4, and Q represents H, —OH,—COOCH₃ phenyl, cyclopropyl, cyclopentyl, cyclohexyl, pyridyl, furyl,thienyl, or oxazolyl. and R₇ is H, or when taken together with thenitrogen atom to which they are attached R₆ and R₇ form apyrrolidine-2-one or pyrrolidine-2,5-dione ring.
 8. A compound asclaimed in claim 1 wherein R₁ is H, F, or Cl; R₂ is H; R₃ is H, F, orCl; Y is —NH—; X is a bond; and R₄ represents —C(═O)NHR₆, —NR₇C(═O)R₆,—NR₇C(═O)OR₆ or —NHC(═O)NHR₆ wherein: R₆ is H or a radical of formula-Alk_(b)-Q wherein b is 0 or 1 and Alk is a —(CH₂)_(n)—,—CH((CH₂)_(m)CH₃)(CH₂)_(n)—, —CH((CH₂)_(m)CH₃)((CH₂)_(p)CH₃)(CH₂)_(n)—,—(CH₂)_(n)—O—(CH₂)_(m)—, or —(CH₂)_(n)—O—(CH₂)_(n)—O—(CH₂)_(m)—, radicalwhere n is 1, 2, 3 or 4 and m and p are independently 0, 1, 2, 3 or 4,and Q represents H, —OH, —COOCH₃ phenyl, cyclopropyl, cyclopentyl,cyclohexyl, pyridyl, furyl, thienyl, or oxazolyl. and R₇ is H, or whentaken together with the nitrogen atom to which they are attached R₆ andR₇ form a pyrrolidine-2-one or pyrrolidine-2,5-dione ring.
 9. A compoundas claimed in claim 1 wherein R₁ is F, R₂ is H or cyclopropyl, R₃ is H,X is a bond, and R₄ is —C(═O)NHR₆, —NRHC(═O)R₆, or —NHC(═O)NHR₆. 10.N-(3-Dimethylaminopropyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzamide,or pharmaceutically or veterinarily acceptable salt thereof.
 11. Acompound as claimed in any of claims 1 to 10 for use in the treatment ofconditions which benefit from immunomodulation.
 12. The use of acompound as claimed in any of claims 1 to 10 in the manufacture of amedicament for the treatment of conditions which benefit fromimmunomodulation.
 13. A method of immunomodulation in humans andnon-human primates, comprising administration to a subject in need ofsuch treatment an immunomodulatory effective dose of a compound asclaimed in any of claims 1 to
 10. 14. A pharmaceutical or veterinarycomposition comprising a compound as claimed in any of claims 1 to 10together with a pharmaceutically or veterinarily acceptable excipient orcarrier.